.'J
WORLD INTELLECTUAL PROPERTY ORGANIZATrON
International Bureau
INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(51) International Patent Classification ^ :
C12Q mo, GOIN 27/327, C12M 1/40,
C12Q 1/54, 1/26
Al
(11) International Publication Number: WO 99/58709
(43) IntematioDal Pablication Date: 18 November 1999 (18.1 1.99)
(21) International Application Number: PCr/GB99/01 424
(22) International Filing Date: 6 May 1999 (06.05.99)
(30) Priority Data:
9809963.3
8 May 1998 (08.05.98)
GB
(71) Applicant (for ail desisnated States except US)t ABBOTT
LABORATORIES [USAJS]; 100 Abbott Park Road, Abbott
Park, IL 60064-3500 (VS).
(72) Inventors; and
(75) Inventors/AppUcants (for US only): STEWART, Alan, An-
drew [GB/GB]; 6 Meadow Lane, Pangboume, Reading,
Berkshire RG8 7NB (GB). SCOTT, Steven [GB/GB]; Ben-
haven, Horn Lane, East Hendred, Oxfonlshire 0X12 8LD
(GB).
(74) Agent: HOWE, Steven; Lloyd Wise, Tregcar & Co., Com-
monwealth House, 1-19 New Oxford Street, London WCl A
ILW (GB).
(81) Designated States: AU. BR. CA, JP. MX, US, European patent
(AT, BE, CH, CY, DE. DK, ES, FI, FR, GB, GR. IE, FT,
LU, MC, NL» FT, SB).
Published
With international search report.
(54)Tiae: TEST STRIP
(57) Abstract
An improved disposable test strip for use in amperometric mea-
surement of analytes in complex liquid media, such as blood, which
has three or more dectrodcs has been developed. This strip is designed
so that different electrical potentials can be maintained between a com-
mon pseudo reference^counter electrode and each of the otl^r electrodes
vspom the imposition of a common potential by an amperometric meter.
This capability is imparted to the test strip by providing different cir-
cuit resistances for each of these other electrodes^ The test strip can be
utilized to measure a single analyte such as ghicose witti a background
compensation via a Mummy** dectrode or it can be used to measure the
concentratioD of multiple anafytes.
FOR THE PURPOSES OF INFORMATION ONLY
Codes used to identify States party to the PCT on Qie front pages of pamphlets publishing Intemationai plications under the PCT,
Albania
ES
Spain
LS
Lesotho
SI
AM
Armeoia
n
inland
LT
. Utfanania
SK
AT
Austria
FR
Ftance
LV
Lmembomg
SN
AU
Australia
GA
Gabon
LV
Latvia ^ .
sz
AZ
AzeibaijaD
GB
United Kingdom
MC
Monaco
TD
BA
Bosnia and Herz^ovina
GB
Oeofgit
MD
Republic of MoUovft .
Madagascar
TG
BB
Baitados
GH
Ghana
MG
XI
MS
Belgium
GN
Gninea
MK .
The fonxr Yngosiiv
TM
BT
BmUna Faso
GR
Giecoe
Repnblic of Macedonia
TR
BG
Bn]£8ria
HU
Hnngaiy
ML
Mali
Tt
BJ
Benin
IB
frdand
MN
Moogolh
UA
BR
Brazil
IL
brad
MR
Msnitaiiift
UG
BY
BelarBS
IS
Iceland
MW
Malawi
US
CA
Canada .
IT
Italy
MX
Mexico
uz
CF
Central Afrkan Republic
JP
Jipan
NB
Niger
VN
CG
Congo
KS
Kenya
NL
Netheilands
YU
CH
SwUwIand
KG
Kyigyzitan
NO
Norway
ZW
O
COiedlvoite
KP
Democratic People**
NZ
New Zealand
CM
OuneiDon
RqnUie of Korea
PL
Poland
CN
Chfoa
KR
RqmbHe of Korea
PT
IVHtugal
CU
Cuba
KZ
Kazakstan
RO
Romania
CZ
Czech RcpobUe
LC
Saint Lucia
RU
Rnssian IVderatfop
DB
Gcrroanry
U
SD
Sudan
DK
Denmailc
LK
SH Lanka
SB
Sweden
EE
Estonia
LR
Liboia
SG
Singapore
Slovenfai
SiovdJa
Senegal
SwarOaod '
Chad
Togo
Tlti.pkistan
T\ukiueuist8tt
Tuffcey
^^inidad and Tobago
tntnfin
Uganda
United States of America
UzbcMttan
Viet Nam
Yugoslai^
wo 99/58709
PCT/CfB99/01424
TEST STRIP
5
The measurement of analytes such as glucose in complex liquid media
such as human blood by amperometric methods using disposable test strips
has become widely used and is currently employed in a number of
commercial products. In certain configurations it is advantageous to
10 improve the si^l to noise ratio by employing a three electrode system in
wfaidi one electrode serves as a pseudo reference/counter electrode to
establish a reference potential. Typically this is a silver/silver chloride
electrode. A second, woiidng electrode is coated with an enzyme which
promotes an oxidation or a reduction reaction with the intended analyte and
15 a mediator which transfers electrons between the enzyme and the electrode.
The third "dummy" electrode is coated with the mediator but not the enzyme
and it provides a measure of the current which arises from other than the
oxidation reduction reaction involving the target analyte. An example of
such a system is described in U.S. Patent No. 5,628,980 to Carter, et al.
1
\yo 99/58709 PCT/GB99/01424
(incorporated by reference herein) and is utilized in the Medi Sense QID ^
glucose meter.
The three electrode system provides a good way to isolate the current
which arises from the oxidation reduction reaction involving the target
5 analyte such as glucose but it also imposes a higher current load on the
pseudo reference/counter electrode. In some testing envirorraients such as
glucose meters used by diabetics iii their homes it is impractical or
impossible to pretreat the samples to remove possible interferants. Thus
with home use glucose meters the diabetic simply applies a sample of whole
10 blood. Whole blood typically contains a niimber of electrochemically active
species whose concentration may vary from person to person or even from
sample to sample from the same individual. The dummy electrode provides
a measure of current arising from the presence of these interfmnts thus
allowing a normalization which ranoves their contribution to the current
15 measured at the working electrode. However, in such a three electrode
configuration the current seen by the pseudo reference/counter electrode
includes contributions from both the working electrode and the dummy
' electrode. Thus in some cases the pseudo reference/county jelectrode sees a
significantly greater current than it would in a two electrode configuration.
wo 99/58709 PCT/GB99/01424
The pseudo reference/counter electrode in siich a configuration is, in
fact, serving two roles which can be inconsistent if the current it sees
becomes too great. It serves, on the one hand, to provide a constant half-cell
potential, i.e. a reference potential and, on the other hand, it also serves as a
counter electrode balancing the electron transfer occurring at the working
and dummy electrodes. For instance, in a typical glucose meter,mediator is
becoming oxidized at the working and dummy electrodes so a reduction
reaction needs to occur at the pseudo reference/counter electrode to balance
the electron transfer. With the typical Ag/AgCl pseudo reference/counter
electrode this involves the reduction of silver ions thus consuminig (or
reducing) silver chloride. If too much silver chloride is consumed the
pseudo reference/counter electrode can no longer serve its fimction of
providing a source of constant half-cell potential. In other words, the
potential difference between the two electrode reactions such as the
oxidation of a mediator at the working electrode and the reduction of silver
at the pseudo reference/counter electrode will actually shift as the reaction
proceeds.
One approach is to redesign the pseudo reference/counter electrode to
handle higher current loads without displaying a significant shift in half-cell
potential. This would normally mean increasing the size or silver
wo 99/58709 PCT/GB99/01424
concentration of the pseudo reference/counter electrode relative to the
working and dummy electrodes. It is difficult to further reduce the size of
the working electrode because its size has already been minimized. It is
limited by the economically acceptable procedures for reproducibly
manufacturing millions of such disposable test strips. On the other hand,
increasing the size or silver concaitration of the pseudo reference/counter
electrode would significantly increase the cost of such three electrode
disposable strips because silver is the most expensive material used in the
construction of such strips.
Therefore, there is a need for three electrode disposable test strips for
use in amperometric systems whose cost is comparable to two electrode test
strips and yet have pseudo reference/counter electrodes with about the same
stability as in the two electrode test strips.
It has been discovered that the current load on the pseudo
reference/counter counter electrode in a disposable test strip/or tise in
amperometric measurements with a three electrode system can be decreased
and therefore its half cell potential better stabilized by increasing the
wo 99/58709 PCT/GB99/01424
resistance of the dummy electrode. This allows three electrode test strips to
give better perfonnahce without changing the operating characteristics of the
meters in which they are used.
Increasing the resistance of the dummy electrode not only reduces the
total current passing through the pseudo reference/counter electrode but it
also changes the potential at the dummy electrode's interface with the
sample. Thus it is possible to have a three electrode system which can
simultaneously measure the concentration of two analytes. The effective
potential at the "dummy* electrode with the higher total resistance can be
adjusted to be too lo>y to effect an oxidation reduction reaction indicative of
the concentration of one of the two target analytes.
It is preferred to have the resistance of the dummy electrode be at
least 1000 ohms greater than that of the working electrode and it is
especially preferred that the resistance differential be at least about 4000
ohms.'
It is also preferred that the resistance of the dummy electrode be
increased by putting a resistance in series with the active electrode surface of
this electrode. Thus both the area and nature of the active sur&ce of the
dummy electrode are kept similar or identical to that of the working
electrode. This can readily be achieved by increasing the resistance of the
I
wo 99/58709 PCT/GB99yO]424
conductive track which connects the active electrode surface to the meter
. which applies the potential and measures the resulting current. In the typical
disposable strip for amperometric analyte measurement three electrode
surfaces are present on one end of an elongated flat strip and three contact
5 pads, one for each of the electrode surfaces, are present on the other end of
the strip. Each electrode surface is connected to its contact pad by a
conductive track. The contact pads serve as the means to estabhsh electrical
contact between the strip and the meter which applies the potential and
measures the resultant current The conductive tracks are typically covered
10 by an insulating layer to prevent any short circuits between them.
It is particularly preferred to increase the resistance of the conductive
track of the dummy electrode by narrowing its width. If this conductive
track is made of the same material as the working electrode^s conductive
track and has about the same thickness as the conductive track of the
15 working electrode it will have a higher resistance. Such a mechanism of
increasing resistance is particularly easy to implement
manufacturing.
6
wo 99/58709
An example
in accordance with
Figs, la and lb are schematic diagrams depicting the conductive
layers of electrodes of disposable test sUips having dummy/second working
electrodes with narrowed conductive layers.
Fig. 2 is a schematic diagraiii depicting the conductive layers of
electrodes of a control disposable test strip ;
Fig. 3 is an Kcploded view of a disposable lest strip ;
Fig. 4 is a perspective view of the assembled strip of Fig. 3 ; and
Fig, 5 is a sraies of plots of current in microamps versus time in
seconds for a working electrode subjected to an initial potential of 4G0
millivolts in the presence of a glucose containing sanq?le for various dummy
electrode configurations.
The three electrode disposable test strip for the amperpinetric measufement
of analytes in complex liquid media is optimized to improve th^ si^l to
5 noise ratio without imposing an excessive current load on the
reference/counter electrode by increasing the resistance of the dummy
electrode, i.e. the electrode which carries the electrochemical mediator also
PCT/GB99/01424
of the present Invention will be described
the acconpanying drawings, in which:
wo 99/58709 PCT/GB99/01424
Utilized at the working electrode but which has no enzyme or other reactant
selected to engage the analyte in an oxidation reduction reaction. A typical
environment for the application of this concept is the three electrode test
strip described in U.S. Patent No. 5,628,890 for the deteraiination of glucose
5 in whole blood samples.
Such a test strip is typically constructed of an elongated strip of a rigid
electrically non-conducting material such as plastic. Suitable plastics
include PVC, polycarbonate or polyester. Three conductive tracks are laid
on this strip so as to establish independent conductive paths from one end to
10 the other. Each track terminates at the end adapted to be proximate to the
meter used to apply electrical potential and measure the resulting currents
with a contact pad that interfaces with the meter. At the distal end of the
strip each track tenjiinates in an electnxie adapted to contact the complex
liquid medium which carries the analyte to be measured A typical medium
15 is whole himaan blood and a typical analyte is glucose.
The working electrode is a pad which is coated with both a substance
del^igned to engage the target analyte in an oxidation-reduction reaction and
a i£ediator adapted to transfer electrons between
reduction reaction. A typical substance is an enzyme adapted to promote the
20 oxidation of glucose, such as glucose oxidase, and thet mediator is a
8
WO99/OT709 PCT/GB99/0I424
compound which readily transfers electrons from the oxidation reduction
reaction to the pad, such as a ferrocene derivative.
The "dummy" electrode is a pad which preferably has the same
surface area as the working electrode and is coated with the siame amount of
the same mediator as the working electrode. The concept is to provide an
environment in the immediate vicinity of this "dummy" electrode which is
essentially identical to that of the working electrode except for the
substance, typically an enzyme, adapted to react with the target analyte.
Then the spurious electrochemical reactions which might occur at the :
working electrode giving rise to noise are just as Ukely to occur at the
"dummy" electrode. Thus the signal arising from such spurious reactions
can be determined by measurement at the "dmmny" electrode and subtracted
from the total signal measured at the working eliectrode. This provides an
improved signal to noise ratio.
The pseudo reference/counter electrode is a pad with a material such
as silver/silver chloride which has both the oxidize^d and reduced form of a
species to provide an essentially constant half-cell potential. So long as the
relative proportions of the reduced and oxidized form of this species siich as
silver and silver chloride are not substantially changed the half-cell potential
of this electrochaiiical couple will remain relatively constant. This
wo 99/58709
PCT/GB99/01424
facilitates being able to maintain a known constant oxidation or reduction
potential at the working electrode. This allows a production batch of
disposable test strips to have a common calibration.
In the typical situation the disposable strips are utilized with a meter
which functions to correlate the amount of current observed upon the
application of an extanal potential to the contact pads of the disposable strip
to the amount of analyte present. This meter is designed to assume certain
electrical characteristics will be observed upon the appUcation of this
extemal potential. One such assumption is that the amount of current
observed vwU decrease monotonically with time! If the current does not
decay ia the cTipected manner the meter is programmed to abort the test. If
the half-cell potential of the pseudo reference/counter electrode such as a
silver/silver chloride electrode shifts the current characteristics may indeed
fail to meet the ojpectations programmed iiito the meter causing an aborted
/test. . • ,
For escample the half^dl potaitial of the silver/silver
electrode will shilt if the proportion of silver tp silver chlcaide is changed.
As rairrent flows through this electrode silver is either redticed or oxidized,
depending on the nature of the reaction occurring at the working electrode.
In the typical meter for sensing glucose concentration glucose is oxidized at
10
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the working electrode reducing the mediator. The mediator then transfers
the electron or electrons it has gained in this reduction reaction to its
electrode pad. These electrons are then taken up at the pseudo
reference/counter electrode. In the typical case this is a silver/silver chloride
electrode and the electrons are taken up by the reduction of silver ions
transforming silver chloride to silver metal.
If a sufficient amount of current passes through such a pseudo
reference/counter electrode the proportion of silver to silver chloride will
change enough to cause a noticeable change in the half-cell potential of this
electrode. If this change becomes large enough the current at the working
electrode may no longer decay monotonically. This in turn will cause the
meter to sense an error condition and abort the test *
The current at the working electrode arises from the oxidation
reductioii reactioii involving the target analyte and the subsequent transfer of
electrons by the mediator. In the typical glucose meter glucose is oxidized
by glucose oxidase and the mediator, for instance a ferrocene derivative,
then transfers the electrons liberated by the oxidation of the glucose to its
electrode pad. In detail the glucose oxidase becomes reduced by oxidizing
the glucose in the sample which is exposed to the disposable test strip and
then is reoxidized by reducing the mediator. The mediator in tum becomes
II
wo 99/58709
PCT/GB99/01424
reoxidized by transferring electrons through its electrode pad to the circuit
with the pseudo reference/counter electrode. Normally the current arising
from this transfer decays monotonically in accordance with the Cottrell
equation as the mediator in reasonable diffusion distance to the electrode pad
5 which was reduced by reaction with glucose oxidase is reoxidized
However, this behavior is dependent upon the potential at the working
electrode being held at or above a certain potential relative to the pseudo
reference/counter electrode. If the potential at this pseudo reference/counter
electrode shifts, the behavior at the working electrode may no longer follow
10 this pattern.
The disposable strips are typically designed so that the pseudo
reference/counter electrode does not undergo such a potential shift. . For
instance this electrode can be made large enough that the current gentrated
by the analyte concentrations typically encountered does not consume
1 5 enough silver ions to cause such a shift.
The use of a third, "dumm/* electrode, however, imposes an
additional current load on the pseudo reference/counter electrode. In the
typical glucose meter where an oxidation reaction occurs at tiie working
electrode, the reduction reaction occurring at the pseudo reference/counter
20 electrode must balance not only the oxidation reaction at the working
12
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electrode but also any oxidation reaction occuning at the "dummy"
electrode. This additional burden may be sufficient to shift the half-cell
potential of the pseudo reference/counter electrode out of its design range.
This is a particular problem in glucose meters which utilize an
initially reduced mediator such as a ferrocene derivative. In such a meter
there is an initial high current load as the mediator is oxidized at both the
working and "dummy" electrodes. If there is also a high level of glucose in
the sample beii^ tested, there will also be a fairly high current load from the
reoxidation of mediator initially reduced as a result of the oxidation of the
glucose. The combined cunent load has a tendency to adversely effect the
half-cell potential of the pseudo reference/counter electrode.
'The total cuirent load on the pseudo reference/counter electrode can
be reduced by increasing the resistance in the overall circuit. However, it is
impractical to change the resistance in the circuit involving the working
electrode. The metars used with the disposable test strips of present concern
are calibrated to correlate the level of cunent in the working electrode circuit
after sometime period or ovct some fixed time interval after exposure of the
test strip to the sample to the concentration of target analyte. Then the
meters are distributed to a large number of users who expect to use the
meters with the disposable test strips for a number of years. Thus it is
13
wo 99/58709 PCT/GB99/01424
electrode circuit increases, i.e. there are few species to support electron
transfer. Thus although there will always be a fixed difference in resistance
between the working and "dummy'' electrodes circuits the percentage
difference will decrease as the effective resistance in the working electrode
circuit increases.
In an alternative embodiment, the three electrode arrangement is used
to simultaneously measure the concentration of two analytes. In this case
there are two working electrodes and one pseudo reference/counter
electrode. The first working electrode is designed to operate with a first
substance that engages one of the target analytes in an oxidation reduction
reaction at a relatively low potential. The second working electrode is
designed to operate with a second substance that engages the other target
analyte in an oxidation reduction reaction only at a higher potential. For
ease in manufacturing both working electrodes are typically coated with
both substances and appropriate mediators. However, the test strip is
designed so that the second substance which is coated on the first working
electrode remains inactive. In particular, the electrical resistance in the
circuit path firom the contact pad connected to the first working electrode
through the first working electrode is significantly greatly than the electrical
resistance in the circuit path from the contact pad connected to the second
IS
WOW/58709 PCT/GB99/01424
impractical to make any change in such test sti-ips which would require a
corresponding change in the meter with which they aire used.
It has, however, been found that the resistance in the "dummy"
electrode circuit can be increased without adversely effecting the interaction
between the disposable test strip and its meter. The function of the
"dummy" electrode is to allow subtraction from the total signal or current at
the working electrode of that portion attributable to superious oxidation-
reduction reactions with species in the complex liquid medium other than the
target ^lyte. This subtraction is only of concoii at the time or over the
interval during Which the current at the working electrode is measured for
correlation to the arialyte concentration. Typically such measurements are
made after the resistance of the overall system is comparatively high after
most of the oxidation at the working electrode has already occurred. It has
been discovered that at this point the difference in electrochemical
environments at the working and "dummy" electrodes is insufficient to
adversely effect tiie function of the dummy electrode.
The relative diffdrence in electrocheniical mvironmrn^
working electrode aihda "dummy*' electrode with added resistance does tend
to decrease as a test cycle proceeds As the rnediator subject to reoxidation
at the working electrode decreases the effective resistance in the working
14
wo 99/58709 PCT/GB99/01424
working electrode through the working electrode. Thus when a certain
electrical potential is applied to the contact pads of both electrodes relative
to the pseudo reference/counter electrode, the effective potential at the first
working electrode is less than that at the second working elec^ode, some of
the potential drop having been expended traversing the higher circuit
resistance.
The two analyte embodiment is applied to the simultaneous
measurement of ketones and glucose by utiUzing an enzyme mediator
^tem for the ketones which operates at +200mV and an enzyme mediator
system for the glucose which operates at -HCKtaiv. In particular, hydroxy
butyrate dehydrogenase (HBDH) with a nicotinamide adenine dinucleotide
(NADH) cofector and a 1,10-phenanthroline quinone (1,10 PQ) mediator is
used for Ae ketones and glucose oxidase with a ferrocene derivative
mediator is used for the glucose.
The low operating potential of the HBDH/NADH/1 , 1 0 PQ system is a
significant advantage for an analyte like ketones which has a limited linear
resjponse range. In the case of ketones a linear response i? typically expected
only over a range of between about 0 and 8 milli Molar. By pperating at a
low potential interferience firom other species which might undergo an
oxidation reduction reaction at a higher potential is avoided. In other words.
16
wo 99/58709 PCT/GB99/01424
the probability that another cheiiiical species in the sainple might become
oxidized and deUver electrons to the first working electrode thus making a
superious contribution to the current sensed at this electrode is minimized.
The potential at the first working electrode is adjusted so that upon the
application of a 400mV potential between the second working electrode and
the reference/counter electrode the potential between this first working
electrode and the reference/counter electrode is 200mV. This adjustment is
effected by increasing the resistance of the circuit path involving this
electrode relative to that involving the second working electrode by an
appropriate amount in one of the ways discussed hereinabove.
The current sensed at the first working electrode is the result of the
oxidation of ketones while that sensed at the second working electrode is the
result of the oxidation of both ketones and glucose. The amount of current
at each electrode can then be employed m a skaple simultaneous equation to
determine the concentration of ketones and glucose in the same sample,
It is, of course, possible to coat only the first working electrode with
the ketcmes sensitive chemistry and to coat only the second workiiig
electrode with only the glucose sensitive chemistry. This would be expected
to result in higher mahufacturirig costs. Typically the disppsable test strips
are manufactured by a series of printmg steps so that applying different
17
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chemistries to each working electrode would require additional printing
steps.
A particular application of the concept of a high resistance dummy
electrode to the measurement of glucose is illustrated in Figures I through 5.
In the strips illustrated, the working electrode and the dummy electrode each
had a surface area of 6.6 1 2 square millimeters while the pseudo
reference/counter electrode had a surface area of 4. 18 square millinieters.
The conductive tracks which connect the contact pads to the electrode pads
are in most eases 0.801 niillimeters. In two cases the conductive track
associated with the dunmiy electrode was narrowed to 0.5 10 millimeters and
0.305 inillimeters, as illustrated in Figures la and lb.
Two different conductive layer prints are illustrated in Figs, la (Track
A) and lb (Track B). A control conductive layer print, in which the working
and dunmiy electrodes have the same resistance, is shown in Fig. 2.
Referring to Figs. la,-lb and 2; the electrode configuration on the scdsoi
strips has three printed layers of electrically conducting carbon ink 2. The
layers define the positions of the pseudo reference/counter electrode 4, the
working elecirode 5; the dtunmy electrode 5a and electrical contacts 3.
Referring to Fig. 2, working electrode 5 has a track width 1 6 that is
equal to track width 16a of dunmiy electrode 5a. Equal track widths 16 and
IS
wo 99/58709 . FCT/GB99/01424
16a give the working electrode and dummy electrode equal resistances.
Referring to Figs, la and lb. track widths 16b and 16c of dummy electrode
5a are narrower than track width 1 6a of the control in Fig. 2. The
conductive layer of dummy electrode 5a is narrowed in order to increase the
resistance of the dummy electrode relative to the working electrode
resistance. Track width 16c is smaller than track width 16b. Thus, the
resistance of dummy electrode 5a in Track A (Fig. la) is greater than the
resistance of dummy electrode 5a in Track B (Fig. lb).
The composition of the conductive layers can also affect the resistance
of the electrodes. Generally, the conductive layers of the electrodes are
printed at the same time with the same ink. The conductive layers can be
printed with a low carbon-content ink or a high carbon-content ink. Low
carbon-content had a carbon content of between 30 and 3 1 weight percent
and a resin content of between 7 and 9 weight percent. The high carbon-
content ink has a carbon content of between 42 aiid 45 weight percent, and a
resin content of between 7 and 9 weight percoit:
A suitable electrode sensor strip is illustrated in Figs. 3 and 4.
Referring to Figs. 3 and 4, the electrode support 1 , an elongated strip of
plastic material (e.g., PVC, polycarbonate, or polyester) supports three
19
wo 99/58709 PCT/GB99/01424
printed tracks of electrically conducting carbon ink 2. These printed tracks
define the positions of the pseudo reference/counter electrode 4, of the
working electrode 5, of the dummy electrode 5a, and of the electrical
contacts 3 that are inserted into an appropriate measurement device (not
shown). The conductive layer of dummy electrode 5a is narrowed in order
to increase the resistance of the dummy electrode relative to the working
electrode.
The elongated portions of the conductive tracks are each overlaid with
silver/silver chloride particle tracks 6a and 6b, with the enlarged exposed
area ovCTlying 4, and 6b and 4 together forming the pseudo
reference/counter electrode. The conductive track or layer for dummy
electrode 5a is not overlaid with silvar/silver chloride. .This further increases
the resistance of the dmnmy electrode. The conductive tracks are further
overlaid with a layer of hydrophobic electrically insulating material 7 that
leaves exposed only the positions of the pseudo reference/counter electrode,
the working electrode and the dununy electrode, and the con^^ This
hydrophobic insulating inaterial prevents short circuits. Because this
iiistilating material is hydrophobic, it can confine the sample-to the exposed
electrodes. A preferred insulating material is available as POLYPLAST°
(Sericol Ltd., Broadstairs, Kent, UK).
30
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The working electrode working area 8 is formed from an ink that
includes a mixture of an enzyme, a mediator/and a conductive material.
The dummy electrode working area is formed from ink that includes a
mixture ofa mediator and a conductive material without enzyme. The
5 respective inks are applied to the positions 5 and 5a of carbon tracks 2 as
discrete areas of fixed length. Alternatively, instead of an enzyme, electrode
layer 8 can contain a substrate catalytically reactive with an enzyme to be
assayed. The conductive material in a preferred embodiment includes
particulate carbon having the redox mediator adsorbed thereon.
10
A printing ink is formed as an aqueous solution of the conductor and
adsorbed redox mediator. For the working electrode, it also includes the
enzyme or. alteniatively, a substrate. When the analyte to be measured is
blood glucose, the en^e is preferably glucose'oxidase, and the redox
15 mediator is a ferrocoie diaivative.
The ink can be screen printed: The ink can include a polysaccharide
(e.g., a guar giiih or an alginate), a Iqrdrdlyzed gelatin, an enzyme stabilizer
(e!g., glutamate or trehalose), a film-forming polymer (e.g., a polyvinyl
alcohol), a conductive filler (e.g., carbon), ia redox mediator (e.g;, ferrocene
20 or a ferrocene derivative), a defoaniing agent, a buffer, and an enzyme or a
21
wo 99/58709 PCT/GB99/01424
subistrate. The ink printed on a dummy electrode lacks the enzyme or the
. substrate.
The pseudp reference/counter electrode 6b is situated relative to the
working electrode 8 and dummy electrode 8a such that it is in a non-ideal
5 position for efficient electrochemical function. The electrodes are arranged
not to rniniinize the effect of the resistance of the solution on the overall
resistance of the circuit (as is conventional), Positioning the pseudo
reference/counter electrode downstream of the working electrode has the
advantage of preventing completion of a circuit (and thus detection of a
10 response) before the working electrode has been completely covered by
sample.
The electrode area is overlaid by a fine grade mesh 9. This mesh
protects the printed components from physical d£umage. It also helps the
sample to wet the pseudo reference/counter electrode and working electrode
15 by reducing the surface tension of the sample, thereby allowing it to spread
evenly over the electrodes. Preferably, this mesh layer extends over the
whole length of the sample path, between and including, the application
point and the electrode: area. Preferably, this.mesh is constructed of finely
woven nyloii strands. Altematiyely, any. woven or non-woven material can
20 be used, provided it does not occlude the surface of the electrode such that
22
wo 99/58709 PCT/CB99/0I424
nonnal difftision is obstructed. The thickness of the mesh is selected so that
the resulting sample depth is sufficiently small to produce a high solution
resistance. Preferably, the fabric is not more than 70 \im in thickness.
Preferably the mesh has a percent open area of about 40 to about 45%, a
mesh count of about 95 to about 1 15 per cm, a fiber diameter of about 20 to
about 40 ^im, and a thickness of from about 40 to about 60 urn. A suitable
mesh is NY'64 HC mesh, available from Sefar (foraierly ZBF), CH-8803,
Ruschlikon, Switzerland.
The mesh can be surfactant coated. This is only necessary if the mesh
material itself is hydrophobic (for example, nylon or polyester), If a
hydrophilic mesh is used, the surfectant coating can be omitted. Any
suitable surfactant can be used to coat the mesh, so long as it allows
adequate even spreading of the sample. A prefeired surfactant is FC 170C
FLUORAD° fluorochemical surfactant (3M, St. Paul, MN). FLUORAD° is
a solution of a fluoroaliphatic oxyethylene adduct, lower polyethylene
glycols, 1 ,4-dioxane, and water. A prefeired surfactant loading for most
applications is from about 15-20 ng/mg of mesh. The prcfetred surfactant
loading will vary depending on the type of mesh and surfectant used and the
sample to be analyzed. It can be detennined empirically by observing fiow
of the sample through the mesh with different levels of surfactant.
23
wo 99/58709
PCT/GB99/01424
A second layer of coarser surfactant coated mesh 10 is applied over
. the first mesh. This second mesh layer controls the influx of the sample ais it
travels firom the application point toward the pseudo reference/counter and
working electrode areas by providing a space into which the displaced air
5 within the sample transfer path can move as the sample moves preferentially
along the lower fine grade mesh layer 9 and partially in mesh layer 10. The
spacing of the larger fibers of the secondary mesh layar, perpendicular to the
direction of sample flow, helps to control the sample flow by presenting
repeated physiical barriers to the movement of the sample as it travels
10 through the transfer path. The regular pattern ofthe mesh fibers ensures that
the sample progresses in stages and that only samples with sufficioit volume
to generate an accurate response are able to pass all the way along the
. pathway and reach the pseudo reference/counter electrode.
15 Preferably, mesh 10 is pfa woven construction, so that it presents a
rejgular repeating pattern of mesh fibers both perpendicular to and parallel to
the longest aspect of the strip. Generally, the second mesh layer should be
substantially thicker than the first mesh, with larger diameter^esh fibers
and largCT apertures between them. The lai^er mesh preferably has a
20 thickness of firom 100 to 1000 |im, with a thickness of from 100 to 150 jmi
24
wo 99^709 • PCr/GB99/01424
being most preferred. A preferred mesh has a percent open area of about 50
to about 55%, a mesh count of from about 45 to about 55 per cm, and a fiber
diameter of from about 55 to about 65 \an. A preferred mesh is NY 1 5 1 HC
mesh, also available from Sefar, CH-8803, Rushchlikon, Switzerland.
5 Mesh 10 is also provided with a coating of a suitable surfactant
(unless the mesh itself is hydrophilic). Preferably, it is the same surfactant
as that on the first mesh layer. The loading of surfactant is lower on mesh
10 than on mesh 9, providing a fiiither barrier to movement of sample past
the transverse fibers of mesh 10. In general, a loading of 1-10 jig/mg of
10 mesh is preferred.
The mesh layers 9 and 10 are held in place by layers of hydrophobic
electrically insulating ink 1 1. These layers can be appUed by screm printing
the ink over a portion of the peripheries of the meshes. Together, the layers
and mesh surround and define a suitable sample transfer path 12 for the
15 sample to travel from the application point at the fiirthest end of the sfrip
towards the working elecfrode and pseudo reference/counter electrode. The
ink impregnates the miesh outside of path 1 2. The insulating material thus
defines sample transfer path 12 by not allowing sample to infiltrate the area
of mesh covered by the layers of insulating material. A preferred insulating
25
wo 99/58709 PCT/GB99/01424
ink Tor impregnating the mesh layers is SERICARD° (Sericol, Ltd.,
Broads tairs, Kent, UK).
The upper part of the electrode is enclosed by a liquid/vapor
impermeable cover membrane 13. This can be a flexible tape made of
polyester or similar material which includes a small aperture 14 to allow
access of the applied sample to the underlying surfactant coated mesh layers.
The impermeable cover membrane encloses the exposed working electrode
and pseudo reference/counter electrode. Thus, it maintains the available
sample space over the electrodes at a fixed height which is equivalent to the
thickness of both mesh layers 9 and 10. This ensures that the solution
resistance is kept at a high level. Any sample thickness up to the maximum
depth of the two mesh layors is adequate in this respect. Aperture 14 is
positioned overlying the furthest end of the open mesh area, remote from the
pseudo reference/counter electrode 6b, such that the exposed area of mesh
beneath the aperture can be used as a point of access or application for the
liquid sainple to be measured. The aperture can be of any suitable size large
enough to allow sufficient volume of sample to pass through to the mesh
layers. It should not be so large as to expose any of the area of ti^^
electrodes. The aperture is formed in the cover membrane by any suitable
method (e.g., die punching). The cover membrane is affixed to the strip
26
wo 99/58709 PCT/GB99/0I424
along a specific section, not including the electrodes, the sample transfer
path or application area, using a suitable method of adhesion. Preferably
this is achieved by coating the underside of a polyester tape with a layer of
hot melt glue which is then heat welded to the electrode surface. The hot
melt glue layer is typically of a coating weight between 10-50 g/m ,
preferably from 20 to 30 g^il Pressure sensitive glues or other equivalent
methods of adhesion may also be used. Care should be taken when the tape
is applied, the heat and pressure applied to the cover membrane can melt the
SERICARD° and can cause it to smear onto adjoining areas.
The upper surface of the cover membrane can also be usefully
provided with a layer of silicone or other hydrophobic coating which helps
to drive the applied sample onto the portion of exposed surfactant coated
mesh at the application point and thus make the application of small
volumes of sample inuch simpler.
In use, a disposable test stirip of the inventirai is connected, via electrode
contacts 3. to a meter (not shown). A sample is applied to aperture 14, and
moves along the saniple transfer path 12. The progress of the sample is
sufficiently impeded by mesh layer 10 to allow the sample to form a uniform
front rather than flowing non-uniformly. Air is displaced thorough the upper
wo 99/58709 PCT/GB99/01424
portion of mesh layer 10 to and through aperture 14. The sample first covers
working electrode 5 in its entirety, and only then approaches and covers
pseudo reference/counter electrode 4. This completes the circuit and causes
a response to be detected by the ineasuring device.
5 The effect of increasing the resistance of a dummy electrode in a
system for measuring glucose in a whole blood sample was electronically
modeled. In particular, Medisense G2a diisposable test strips which utilize
glucose oxidase and a forocene mediator were tested using venous blood
spiked with glucose to a concentration of ISmM. The electronics was used
1 0 to simulate the effect of having a dummy electrode with each of five added
resistances from zero to infinity (no dummy electrode). An initial potoitial
relative to the pseudo reference/counter electrode of 400 mV was imposed
on the working electrode and the current at the working electrode was
monitored over time. The results were reported in Figure 5.
15 Figure 5 illustrates that as the resistance increases so does the current
at tte working electrode. This is an indirect indipation that the half cell
potential of the pseudo jrefCTence/counter electrode is being stabilized. In an
ideal situation the current at the working electrode should be independent of
the resistance of the dummy electrode and should just depend upon the rate
20 at which glucose is oxidized. However, in the real world the extra current
28
wo 99/58709 . PCT/GB99/0I424
load imposed on the pseudo reference/coiinleir electrode by the dummy
electrode does cause an observable shift in the half cell potential of the
pseudo reference/counter electrode. This in turn has an effect upon the
current observed at the working electrode. As the potential difference
5 between the working and pseudo reference/counter electrodes decreases
because of this shift so does the current at the working electrode.
In addition, under some conditions the current decay at the working
electrode departs from the expected model. In particular, it is expected the
cuirent will decrease monotonicly with time and tend to exhibit the behavior
10 predicted by the CottreU equation. However, under certain conditions when
the dummy electrode is imposing a significant current load on the pseudo
reference/counter electrode the cuirent at the working electrode departs from
classical behavior and may actually increase with time over some short time
period. This is clearly illustrated in the lowest most curve of Figure 5,
15 wluch represents a disposable test strip in which there is no resistance
differential between the circuit path involving the working electrode and that
involving the dummy electrode.
The glucose meters with which the disposable test strips of pr^ent
concem are typically used have electronic features designed to detect invalid
20 test results. One of these check features is a monitoring of the current decay
29
wo 99/58709 PCT/GB99yO]424
al the working electrode. If this decay is not monotonic the meter will report
an error condition and abort the test.
Thus increasing the resistance of the dummy electrode has been
shown to be effective in decreasing the likelihood of a non-monotonic
current decay at the working electrode and the consequent abortion of a test.
30
wo 99/58709
PCT/GB99/01424
CLAIMS:
1^ A disposable test strip suitable for attachment
to the signal readout circuitry of a meter which performs
an amperometric test to detect a current representative
of the concentration of an analyte in a complex liquid
medium comprising:
(a) a working electrode which comprises an
electrode pad coated with both a substance designed to
engage said analyte in. an oxidation- redact ion reaction
and a mediator compound which will transfer electrons
between the oxidation-reduction reaction and the
electrode pad;
(b) a dummy electrode which comprises an
electrode pad which is coated with about the same amount
of mediator compound as the working electrode but lacks
the sxibstance which engages the analyte in the oxidation-
reduction reaction;
(c) a pseudo reference/counter electrode which
comprises an electrode pad coated with a material which
contains both the oxidized and reduced form of a chemical
species which is designed to undergo a reduction or
oxidation reaction to balance the opposite reaction at
;the working and dummy electrodes; and
(d) three conductive tracks, each of which
extends from a contact pad adajpted to interface with said
readout circuitry to brie of the electrode pads and which
is in electrical contact with both its contact pad and
its electrode pad;
wherein the electrical resistance in the circuit path
from the contact pad connected to the dummy electrode
through the dummy electrode is significantly greater than
the electrical resistance in the circuit path from the
contact pad connected to the working electrode through,
the working electrode.
31
wo 99/58709
PCT/GB99/01424
2. The disposable test strip of Claim 1 wherein
the greater electrical resistance in the dumrny electrode
circuit is provided by increasing the resistance of the
conductive track connecting the dummy electrode to its
contact pad,
3* The disposable test strip of Claim i or 2,
further comprising an elongate support having a
substantially flat, planar surface arranged to be
releasably attached to the readout circuitry.
4. The disposable test strip of Claim 3 wherein
the three conductive tracks are created by coating
conductive particles on the elongated support.
5. The disposable test strip of Claim 4 wherein
the conductive particles comprise carbon.
6. The disposable test strip of Claim 4 or 5
wherein a greater electrical resistance is imparted to
the conductive track connecting the dummy electrode to
its contact pad by using a smaller volume of conductive
particles in this track as compared to that used in the
conductive track connecting the working electrode to its
contact pad.
7. The disposable test strip of any one of Claims
2 to 6 wherein the conductive track connecting the dummy
electrode to its contact pad is narrower than the
conductive track connecting the working electrode to its
contact pad. ] ^ ' .
8. The disposable test strip of any one of Claims
2 to 7 wherein the conductive track connecting the dummy
electrode to its contact pad is thinner than the
conductive track connecting the working electrode to its
contact pad.
32
PCT/GB99/01424
9: . The disposable test strip of any one of Claims
2 to 8 wherein the conductive track connecting the dummy
electrode to its contact pad hais a different composition
than the conductive track connecting the working
electrode to its contact pad.
10. The disposable test strip of Claim 9 wherein
both the conductive track connected to the dummy
electrode and the conductive track connected to the
working electrode are comprised of carbon particles but
only the latter conductive track is coated with silver.
11. The disposable test strip of any one of Claims
2 to 10 wherein the conductive track connecting the dummy
electrode to its contact pad is longer than the
conductive track connecting the working electrode to its
contact pad.
12. The disposable test strip of any one of the
preceding claims wherein the analyte is glucose and the
substance engaging the analyte in an oxidation reduction
reaction is ah enzyme.
13 . . The disposable test strip of Claim 12 wherein
the enzyme is glucose oxidase!.
14. The disposable test strip of any one of the
preceding claims wherein the mediator is a ferrocene
derivative.
15. The disposable test strip of any one of the
preceding claims wherein said pseudo reference /counter
electrode comprises an electrode pad coated with a
mixture of silver and silver chloride.
16. The disposable test strip of any one of the
preceding claims wherein the electrical resistance in
33
wo 99/58709
PCT/GB99/01424
said dummy electrode circuit is at least 1000 ohms
greater than in said working electrode circuit path.
17. A disposable test strip suitable for attachment
to the signal readout circuitry of a meter which performs
an amperometric, test to detect currents representative of
the concentrations of multiple analytes in a liquid
medium comprising:
(a) a first working electrode which comprises
an electrode pad coated with both a substance designed to
engage one of the multiple analytes in an oxidation-
reduction reaction at a first electrical potential
difference and a mediator compound which will transfer
electrons between its oxidation-reduction reaction and
its electrode pad;
(b) a second working electrode which comprises
an electrode pad which is coated with both a substance
designed to engage another of the multiple analytes in an
oxidation- reduction reaction at a second electrical
potential difference which is significantly greater than
said first electrical potential difference and another
mediator compound which will transfer electrons between
its oxidation-reduction reaction and its electrode pad;
(c) a pseudo reference/counter electrode which
comprises an electrode pad coated with a material which
contains both the oxidized and reduced form of a chemical
species which is designed to undergo a reduction or
oxidation reaction to balamce the opposite reactions at
the first and second working electrodes; and
(d) three .conductive tracks, each^of which
extends from a contact pad intended to interface with
said readout circuitry to one of the electrode pads and
which is in electrical contact with both its contact pad
and its electrode pad;
wherein the electrical resistance in the circuit path
from the contact pad connected to the first working
34
wo 99/58709
PCT/GB99/01424
electrode through the first working electrode is
significantly greater than the electrical resistance in
the circuit path from the contact pad connected to the
second working electrode through the second working
electrode.
18 . The dispoisable test strip of Claim 17 wherein
there are only two working electrodes,
19. The disposable test strip of Claim 17 or 18
wherein the pseudo reference/ counter electrode comprises
an electrode pad coated with a mixture of silver and
silver chloride.
20. ' The disposable test strip of any one of Claims
17 to 19 wherein the first working electrode comprises an
enzyme system adapted to engage ketones and a suitable
mediator and the second working electrode comprises an
enzyme suitable to engage glucose and a suitable
mediator.
21. The disposable test strip of Claim 20 wherein
the first working electrode comprises a HBDH/NADH/1,00 PQ
system and the second working electrode comprises glucose
oxidase and a ferrocene based mediator.
22. The disposable test strip of Claim 21 wherein
the resistance in the first working electrode circuit is
such that when a 400 mV potential exists between the
second working electrode and the pseudo reference/counter
electrode there is a 200 mV potential between the first
working electrode and the pseudo reference/counter
electrode.
23 . A disposable test strip svibstantially as. shown
in or described with respect to Figures la, lb, 3, 4 or 5
oiE the accon^amying drawings.
35
wo 99/58709
PCT/GB99/01424
SUBSntUTE SHEET (RULE 26)
wo 99/58709
PCT/GB99/01424
suBsrrruTE sheet (rule 2B)
wo 99/58709
PCT/GB99/01424
SUBSTITUTE SHEET (RULE 26)
INtERNATlONAL SEARCH REPORT
Ml JoMi Appllettien No -
PCT/GB 99/01424
A. CLASSFICATONOF^SUBJECT MATTER .^^ ^, m ^^^^^i^^
IPC 6 C12Q1/00 G01N27/327 C12M1/40 C12Q1/54 C12Q1/26
According to IntamaHonaj Palent CtassBteaikm (IPC) or to both nattenal daasiffcatten and iPC
a F1EU>S SEARCHED
Minimum documantatkm aeaiched (classiScatlon system loOowed tiy dassiScation symtwts)
IPC 6 C12Q 601N C12M
Documentation searched other than minimum documentation to the extent thai such documents are included In the fields searched
Electronic data 1)880 oonsufted during the international search (name of data base and. where practical, search terms used)
a DOCUMENTS CONSIDERED TO BE RELEVANT
Category <
Citation of document, with Indteatlon. where appropriate, of the relevanl p as sa g es
Relevant to claim No.
PATENT ABSTRACTS OF JAPAN
vol. 097, no. 012,
25 December 1997 (1997-12-25)
& JP 09 201337 A (CASIO COMPUT CO LTD),
5 August 1997 (1997-08-05)
abstract
1.17
US 5 628 890 A (CARTER NIGEL F
13 May 1997 (1997-05-13)
cited In the appllcatton .
the whole docunent
US 5 509 410 A (HILL HUGH A 0
23 April 1996 (1996-04-23)
abstract
ET AL)
ET AL)
1.17
-/-
l=urther documents are istfldin the continuation of box C.
PflSent iutSbjf memtMfB are Bsted In annex.
* Spec i al categoriss of dted documents i
*A* document de&tlng the general state of the artwhtohbnol
considered to be of particular reieivance
earifar document butpubMied on or sftar the htsmaSonal
HBngdate
V document which may throw doubte on piiorty da!m(s)or
wtdch b cSed to eitobfish the pubUcailon date of another
citation orolherspeda! reason (as specified)
V d o c ume nt l el eii tr igto an oral tfaclpeui e^ use^ eodiSritionor
other means
■P* document pubtehed prior to the Intemaliorral f^ng data but
later ttian ttie pcforty date claimed
*r later document pubBshed after the international faing data
or priority date and not InoontBclwBh the appBcation but
cited to understand the principle or theory underlying the
Invention
"X" document of particular lelavanes; the datmad invention
cannot be oonsidsred novel or cannot be considered to
Involve an inventhre step when the document is takenalone
"Y" document of particular relevance; the claimed imrention
cannot be centred to ^ivolve an inventive step when the
document is oomUned wkh one or more other such docth
- merits, such oomlrination being obvious to a person skiOed
in ttie art,
docanent member of the same patent famffy
Data of the actual oomplBllon of the intematioftal eeaioh
2 August 1999
Date of malingof the international search report
09/08/1999
Name and inaffing address of ttie ISA
European Patent Office. P-B. 5810 PatenUaan 2
NL-2280HVF^swqk
Tel. (+31.70) 340-2040. Tx. 31 651 epo nl,
Fax:(-»31.70)34O3018
Author teed officer
Moreno, C
Fom PCmSAaiO (MQoed ihaeO (Jdy 1992)
page 1 of 2
internahonajl search report
Int JonsI Applleation No
PCT/GB 99/01424
C^Conttnuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT
Calegofy* Citation ol docimieiit. wfth lmicatlon.iiv^
Rdovsnt to d^Ri No»
EP 0 593 096 A (MEOISENSE INC)
20 April 1994 (1994-04-20)
the whole document
WO 97 30344 A (SELFCARE INC)
21 August 1997 (1997-08-21)
abstract
1.17
Ftaira PGT/ISAfilO (oMilnnlloii of MOondthMQ UO^ 19881
page 2 of
2
INTERNATIONAL SEARCH REPORT
iiiitoniurtlofi on pfltsnt tunUy nwmbws
M« lemlApplleatlenlto
PCT/6B 99/01424
Patent document
PubBcation
Patent family
Publication
Cfted in search report
date
memt)er(s)
date .
JP 09201337
A
05-08-1997
NONE
US 5628890
A
13-05-1997
CA
2159553 A
30-03-1997
JP
9222411 A
26-08-1997
US 5509410
A
23-04-1996
GB
2154003 A
29-08-1985
US
5727548 A
17-03-1998
AU
572138 B
05-05-1988
CA
1226036 A
25-08-1987
DE
3485554 A
16-04-1992
DE
3486221 D
04-11-1993
DE
3486221 T
27-01-1994
EP
0127958 A
12-12-1984
EP
0351891 A
24-01-1990
EP
0351892 A
nil n^ 4 MAM
24-01-1990
JP
9325127 A
4 ^ 4 n 4 AM<V
16-12-1997
JP
7072727 B
nn nn 4 nM^
02-08-1995
JP
60017344 A
on m 4 nnr
29-01-1985
US
5682884 A
nit 4 4 4 nn*ff '
04-11-1997
US
5820551 A
4 n 4 A 4 MM#%
13-10-1998
AU
569076 B
21-01-1988
AU
2775384 A
08-11-1984
AU
580257 B
12-01-1989
AU
2775484 A
08-11-1984
CA
1219040 A
10-03-1987
CA
1223638 A
30-06-1987
CA
1218704 A
03-03-1987
CA
1220818 A
21-04-1987
EP
0125867 A
21-11-1984
EP
0125136 A
14-11-1984
EP
0125137 A
14-11-1984
EP
0125139 A
^ A 4 4 4 nn 4
14-11-1984
US
4758323 A
19-07-1988
US
4711245 A
MM 4 M 4 M«%4
08-12-1987
EP 0593096
A
20-04-1994
AU
622196 B
02-04-1992
AU
3822089 A
M4 MM 4 MMM
01-02-1990
CA
1313397 A
02-02-1993
EP
0352138 A
M^ M4 4 MMM
. 24-01-1990
JP
2112752 A
25-04-1990
US
5126034 A
: 30-06-1992
UO 9730344
A
21-08-1997
US
5708247 A
13-01-1998
AU
2269297 A
02-09-1997
CA
2245941 A
21-08-1997
EP
0880692 A
02-12-1998
THIS PASi BUNK (uspto)